The instant inventions are generally related to compositions and methods for intracellular delivery of biomolecular agents such as polynucleotides, and more specifically, for intracellular delivery of such agents using polymeric micelles.
Polymeric micelles are known in the art for delivering therapeutics into cells. PCT Patent Application WO 2008/153940 (Hirt et al.) discloses vesicles prepared from amphiphilic segmented copolymers. Kataoka et al. (2005) (“Smart polymeric micelles as nanocarriers for oligosaccharides and siRNA delivery”, Oxford University Press—Nucleic Acids Symposium Series, No. 49, pp. 17-18) disclose various approaches involving polyionic complexes. PCT Application WO 2008/004978 (Yang et al.) discloses homogeneous micelles adapted for delivery of small molecule therapeutics. U.S. Patent Application Publication No. 2005/0070721 (Bae et al.) discloses heterogeneous carriers adapted for delivery of hydrophobic small molecule therapeutics. U.S. Pat. No. 6,210,717 (Choi et al.) discloses heterogeneous carriers comprising polycation-b-polyesters and polysaccharide-conjugated polyesters for transport of nucleic acids into eukaryotic cells. PCT Patent Application WO 2009/004978 (Torchilin et al.) discloses heterogeneous carriers comprising cholesterol-conjugated small interfering ribonucleic acid (siRNA) and polyethyleneglycol-conjugated phosphatidylethanolamine for polynucleotide delivery.
Known approaches for delivering biomolecular agents using micelles have a variety of shortcomings.
Generally, for example, many such approaches are lacking or are inadequate with respect to desirable functionality, especially with respect to achieving intracellular delivery, with respect to delivery of specific classes of biomolecular agents such as polynucleotides, and/or with respect to achieving targeted delivery to specific cells of interest. For example, various known systems have not contemplated, are ineffective, or have other deficiencies for release of an agent from endosomes into the cytoplasm, after the agent enters the cell through endocytosis. Known systems are also lacking or inadequate for certain features important for delivering polynucleotides, such as providing for adequate association of the polynucleotide with the micelle, while avoiding potential toxicities and enzymatic degradation, and while maintaining robust micellic stability. As a non-limiting example, some known approaches include polycationic functionality for ionic association of negatively-charged polynucleic acids; however, if inadequately shielded, polycations can cause toxicity concerns in vivo (e.g., toxicities mediated by non-specific interactions with plasma proteins or circulating cells). Known approaches have incorporated polynucleotides by ionic association into the core of the micelle; however, such approaches can impact micelle stability. Although various known systems have contemplated targeting approaches for specifically-directed cellular uptake (e.g., via receptor-mediated endocytosis), these systems have been ineffective in integrating such functionality into micelles without compromising other required functionality.
Additionally, various known approaches are generally not sufficiently robust to incorporate multiple, complex functional features required for effective intracellular delivery of biomolecular agents such as polynucleotides. For example, many known carrier systems are formed from naturally-occurring moieties such as lipids or phospholipids (e.g., phosphatidylethanolamine), peptidic polymers (e.g., polyhistidine), or polysaccharides, which are typically more susceptible to biological degradation if inadequately protected, or formed from simple homopolymers (e.g., polyesters such as polylactic acid) which offer few design variations for incorporating multiple functional features, for optimization to enhance such features, or for tuning to tailor such features to specific applications of interest.
As a further shortcoming, known systems for intracellular delivery of biomolecular agents such as polynucleotides are not readily manufacturable. Efforts to achieve delivery vehicles which incorporate multiple functional features are hindered by complex and chemically difficult syntheses—e.g., involving multi-way chemical conjugations, which can be particularly difficult to realize in larger scale production.
Hence, there remains a substantial need in the art for improved compositions and methods for intracellular delivery of biomolecular agents such as polynucleotides, and especially, for improved intracellular delivery of such agents using polymeric micelles.